Naegleria fowleri, the causative agent of primary amoebic meningoencephalitis (PAM), requires increased research attention due to its high lethality and the potential for increased incidence as a result of global warming. The aim of this study was to investigate the interactions between N. fowleri and host cells in order to elucidate the mechanisms underlying the pathogenicity of this amoeba. A co-culture system comprising human fibrosarcoma cells was established to study both contact-dependent and contact-independent cytopathogenicity. Proteomic analyses of the amoebas exposed to human cell cultures or passaged through mouse brain were used to identify novel virulence factors. Our results indicate that actin dynamics, regulated by Arp2/3 and Src kinase, play a considerable role in ingestion of host cells by amoebae. We have identified three promising candidate virulence factors, namely lysozyme, cystatin and hemerythrin, which may be critical in facilitating N. fowleri evasion of host defenses, migration to the brain and induction of a lethal infection. Long-term co-culture secretome analysis revealed an increase in protease secretion, which enhances N. fowleri cytopathogenicity. Raman microspectroscopy revealed significant metabolic differences between axenic and brain-isolated amoebae, particularly in lipid storage and utilization. Taken together, our findings provide important new insights into the pathogenic mechanisms of N. fowleri and highlight potential targets for therapeutic intervention against PAM.

• MeSH
• Virulence Factors metabolism   MeSH
• Coculture Techniques MeSH
• Humans MeSH
• Brain * parasitology   metabolism   pathology   MeSH
• Mice MeSH
• Naegleria fowleri * pathogenicity   metabolism   MeSH
• Proteomics MeSH
• Protozoan Proteins metabolism   MeSH
• Central Nervous System Protozoal Infections * parasitology   metabolism   pathology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Virulence Factors MeSH
• Protozoan Proteins MeSH

Seasonally slowly melting mountain snowfields are populated by extremophilic microalgae. In alpine habitats, high-light sensitive, green phytoflagellates are usually observed in subsurface layers deeper in the snowpack under dim conditions, while robust orange to reddish cyst stages can be seen exposed on the surface. In this study, uncommon surface green snow was investigated in the High Tatra Mountains (Slovakia). The monospecific community found in the green surface bloom consisted of vegetative Chloromonas cells (Volvocales, Chlorophyta). Molecular data demonstrated that the field sample and the strain isolated and established from the bloom were conspecific, and they represent a new species, Chloromonas kaweckae sp. nov., which is described based on the morphology of the vegetative cells and asexual reproduction and on molecular analyses of the strain. Cells of C. kaweckae accumulated approximately 50% polyunsaturated fatty acids, which is advantageous at low temperatures. In addition, this new species performed active photosynthesis at temperatures close to the freezing point showed a light compensation point of 126 ± 22 μmol photons · m-2 · s-1 and some signs of photoinhibition at irradiances greater than 600 μmol photons · m-2 · s-1 . These data indicate that the photosynthetic apparatus of C. kaweckae could be regarded as adapted to relatively high light intensities, otherwise unusual for most flagellate stages of snow algae.

• Keywords
• biodiversity, cryoflora, environmental sample, fatty acids, fluorometry, vegetative stages,
• MeSH
• Chlorophyceae * MeSH
• Chlorophyta * physiology   MeSH
• Photosynthesis physiology   MeSH
• Cold Temperature MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Slovakia MeSH

Despite the widespread occurrence of intracellular crystalline inclusions in unicellular eukaryotes, scant attention has been paid to their composition, functions, and evolutionary origins. Using Raman microscopy, we examined >200 species from all major eukaryotic supergroups. We detected cellular crystalline inclusions in 77% species out of which 80% is composed of purines, such as anhydrous guanine (62%), guanine monohydrate (2%), uric acid (12%) and xanthine (4%). Our findings shifts the paradigm assuming predominance of calcite and oxalates. Purine crystals emerge in microorganisms in all habitats, e.g., in freshwater algae, endosymbionts of reef-building corals, deadly parasites, anaerobes in termite guts, or slime molds. Hence, purine biocrystallization is a general and ancestral eukaryotic process likely present in the last eukaryotic common ancestor (LECA) and here we propose two proteins omnipresent in eukaryotes that are likely in charge of their metabolism: hypoxanthine-guanine phosphoribosyl transferase and equilibrative nucleoside transporter. Purine crystalline inclusions are multifunctional structures representing high-capacity and rapid-turnover reserves of nitrogen and optically active elements, e.g., used in light sensing. Thus, we anticipate our work to be a starting point for further studies spanning from cell biology to global ecology, with potential applications in biotechnologies, bio-optics, or in human medicine.

• MeSH
• Biomineralization * MeSH
• Eukaryota * genetics   metabolism   MeSH
• Guanine metabolism   MeSH
• Humans MeSH
• Purines metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Guanine MeSH
• purine MeSH Browser
• Purines MeSH

Photosynthetic energy conversion and the resulting photoautotrophic growth of green algae can only occur in daylight, but DNA replication, nuclear and cellular divisions occur often during the night. With such a light/dark regime, an algal culture becomes synchronized. In this study, using synchronized cultures of the green alga Desmodesmus quadricauda, the dynamics of starch, lipid, polyphosphate, and guanine pools were investigated during the cell cycle by two independent methodologies; conventional biochemical analyzes of cell suspensions and confocal Raman microscopy of single algal cells. Raman microscopy reports not only on mean concentrations, but also on the distribution of pools within cells. This is more sensitive in detecting lipids than biochemical analysis, but both methods-as well as conventional fluorescence microscopy-were comparable in detecting polyphosphates. Discrepancies in the detection of starch by Raman microscopy are discussed. The power of Raman microscopy was proven to be particularly valuable in the detection of guanine, which was traceable by its unique vibrational signature. Guanine microcrystals occurred specifically at around the time of DNA replication and prior to nuclear division. Interestingly, guanine crystals co-localized with polyphosphates in the vicinity of nuclei around the time of nuclear division.

• Keywords
• Desmodesmus quadricauda, cell cycle, confocal Raman microscopy, guanine, lipids, microalgae, polyphosphate, starch,
• MeSH
• Cell Wall chemistry   MeSH
• Cell Cycle * MeSH
• Time Factors MeSH
• Chlorophyta cytology   growth & development   MeSH
• Guanine analysis   MeSH
• Lipid Droplets metabolism   MeSH
• Lipids analysis   MeSH
• Microscopy * MeSH
• Polyphosphates analysis   MeSH
• Spectrum Analysis, Raman * MeSH
• Starch analysis   MeSH
• Cell Size MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• Guanine MeSH
• Lipids MeSH
• Polyphosphates MeSH
• Starch MeSH